Polysaccharide Enzymes
Both prokaryotic and eukaryotic cells use polysaccharide enzymes as a storage reserve. In the prokaryotic cell the primary reserve polysaccharide is glycogen. Although glycogen is similar to the starch found in most vascular plants it exhibits different chain lengths and degrees of polymerization. In many plants, starch is used as the primary reserve polysaccharide. Starch is stored in the various tissues of the starch bearing plant. Starch is made of two components in most instances; one is amylose and one is amylopectin. Amylose is formed as linear glucans and amylopectin is formed as branched chains of glucans. Typical starch has a ratio of 25% amylose to 75% amylopectin. Variations in the amylose to amylopectin ratio in a plant can effect the properties of the starch. Additionally starches from different plants often have different properties. Maize starch and potato starch appear to differ due to the presence or absence of phosphate groups. Certain plants' starch properties differ because of mutations that have been introduced into the plant genome. Mutant starches are well known in maize, rice and peas and the like.
The changes in starch branching or in the ratios of the starch components result in different starch characteristic. One characteristic of starch is the formation of starch granules which are formed particularly in leaves, roots, tubers and seeds. These granules are formed during the starch synthesis process. Certain synthases of starch, particularly granule-bound starch synthase, soluble starch synthases and branching enzymes are proteins that are “encapsulated” within the starch granule when it is formed.
The use of cDNA clones of animal and bacterial glycogen synthases are described in International patent application publication number GB92/101881. The nucleotide and amino acid sequences of glycogen synthase are known from the literature. For example, the nucleotide sequence for the E. coli glgA gene encoding glycogen synthase can be retrieved from the GenBank/EMBL (SWISSPROT) database, accession number J02616 (Kumar et al., 1986, J. Biol. Chem., 261:16256–16259). E. coli glycogen biosynthetic enzyme structural genes were also cloned by Okita et al. (1981, J. Biol. Chem., 256(13):6944–6952). The glycogen synthase glgA structural gene was cloned from Salmonella typhimurium LT2 by Leung et al. (1987, J. Bacteriol., 169(9):4349–4354). The sequences of glycogen synthase from rabbit skeletal muscle (Zhang et al., 1989, FASEB J., 3:2532–2536) and human muscle (Browner et al., 1989, Proc. Natl. Acad. Sci., 86:1443–1447) are also known.
The use of cDNA clones of plant soluble starch synthases has been reported. The amino acid sequences of pea soluble starch synthase isoforms I and II were published by Dry et al. (1991, Plant Journal, 2:193202). The amino acid sequence of rice soluble starch synthase (SSTS) was described by Baba et al. (1993, Plant Physiology,). This last sequence (rice SSTA) incorrectly cites the N-terminal sequence and hence is misleading. Presumably this is because of some extraction error involving a protease degradation or other inherent instability in the extracted enzyme. The correct N-terminal sequence (starting with AELSR SEQ. ID NO:38) is present in what they refer to as the transit peptide sequence of the rice SSTS.
The sequence of maize branching enzyme I was investigated by Baba et al., 1991, BBRC, 181:8794. Starch branching enzyme II from maize endosperm was investigated by Fisher and Shrable (1993, Plant Physiol., 102:10451046). The use of cDNA clones of plant, bacterial and animal branching enzymes have been reported. The nucleotide and amino acid sequences for bacterial branching enzymes (BE) are known from the literature. For example, Kiel et al. cloned the branching enzyme gene glgB from Cyanobacterium synechococcussp PCC7942 (1989, Gene (Amst), 78(1):918) and from Bacillus stearothermophilus (Kiel et al., 1991, Mol. Gen. Genet., 230(12):136–144). The genes glc3 and gha1 of S. cerevisiae are allelic and encode the glycogen branching enzyme (Rowen et al., 1992, Mol. Cell Biol., 12(1):22–29). Matsumomoto et al. investigated glycogen branching enzyme from Neurospora crassa (1990, J. Biochem., 107:118–122). The GenBank/EMBL database also contains sequences for the E. coli glgB gene encoding branching enzyme.
Starch synthase (EC 2.4.1.11) elongates starch molecules and is thought to act on both amylose and amylopectin. Starch synthase (STS) activity can be found associated both with the granule and in the stroma of the plastid. The capacity for starch association of the bound starch synthase enzyme is well known. Various enzymes involved in starch biosynthesis are now known to have differing propensities for binding as described by Mu-Forster et al. (1996, Plant Phys. 111: 821–829). Granule-bound starch synthase (GBSTS) activity is strongly correlated with the product of the waxy gene (Shure et al., 1983, Cell 35: 225–233). The synthesis of amylose in a number of species such as maize, rice and potato has been shown to depend on the expression of this gene (Tsai, 1974, Biochem Gen 11: 83–96; Hovenkamp-Hermelink et al., 1987, Theor. Appl. Gen. 75: 217–221). Visser et al. described the molecular cloning and partial characterization of the gene for granule-bound starch synthase from potato (1989, Plant Sci. 64(2):185192). Visser et al. have also described the inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs (1991, Mol. Gen. Genet. 225(2):289296).
The other STS enzymes have become known as soluble starch synthases, following the pioneering work of Frydman and Cardini (Frydman and Cardini, 1964, Biochem. Biophys. Res. Communications 17: 407–411). Recently, the appropriateness of the term “soluble” has become questionable in light of discoveries that these enzymes are associated with the granule as well as being present in the soluble phase (Denyer et al., 1993, Plant J. 4: 191–198; Denyer et al., 1995, Planta 97: 57–62; Mu-Forster et al., 1996, Plant Physiol. 111: 821–829). It is generally believed that the biosynthesis of amylopectin involves the interaction of soluble starch synthases and starch branching enzymes. Different isoforms of soluble starch synthase have been identified and cloned in pea (Denyer and Smith, 1992, Planta 186: 609–617; Dry et al., 1992, Plant Journal, 2: 193–202), potato (Edwards et al., 1995, Plant Physiol. 112: 89–97; Marshall et al., 1996, Plant Cell 8: 1121–1135) and in rice (Baba et al., 1993, Plant Physiol. 103: 565–573), while barley appears to contain multiple isoforms, some of which are associated with starch branching enzyme (Tyynela and Schulman, 1994, Physiol. Plantarum 89: 835–841). A common characteristic of STS clones is the presence of a KXGGLGDV (SEQ. ID NO:39) consensus sequence which is believed to be the ADP-Glc binding site of the enzyme (Furukawa et al., 1990, J. Biol. Chem. 265: 2086–2090; Furukawa et al., 1993, J. Biol. Chem. 268:23837–23842).
In maize, two soluble forms of STS, known as isoforms I and II, have been identified (Macdonald and Preiss, 1983, Plant Physiol. 73: 175–178; Boyer and Preiss, 1978, Carb. Res. 61: 321–334; Pollock and Preiss, 1980, Arch Biochem. Biophys. 204: 578–588; Macdonald and Preiss, 1985 Plant Physiol. 78: 849–852; Dang and Boyer, 1988, Phytochemistry 27: 1255–1259; Mu et al., 1994, Plant J. 6: 151–159), but neither of these has been cloned. STSI activity of maize endosperm was recently correlated with a 76-kDa polypeptide found in both soluble and granule-associated fractions (Mu et al., 1994, Plant J. 6: 151–159). The polypeptide identity of STSII remains unknown. STSI and II exhibit different enzymological characteristics. STSI exhibits primer-independent activity whereas STSII requires glycogen primer to catalyze glucosyl transfer. Soluble starch synthases have been reported to have a high flux control coefficient for starch deposition (Jenner et al., 1993, Aust. J. Plant Physiol. 22: 703–709; Keeling et al., 1993, Planta 191: 342–348) and to have unusual kinetic properties at elevated temperatures (Keeling et al., 1995, Aust. J. Plant Physiol. 21807–827). The respective isoforms in maize exhibit significant differences in both temperature optima and stability.
Plant starch synthase (and E. coli glycogen synthase) sequences include the sequence KTGGL (SEQ ID NO:40) which is known to be the adenosine diphosphate glucose (ADPG) binding domain. The genes for any such starch synthase protein may be used in constructs according to this invention.
Branching enzyme [α1,4Dglucan: α1,4Dglucan 6D(α1,4Dglucano) transferase (E.C. 2.4.1.18)], sometimes called Q-enzyme, converts amylose to amylopectin. A segment of a α1,4Dglucan chain is transferred to a primary hydroxyl group in a similar glucan chain.
Bacterial branching enzyme genes and plant sequences have been reported (rice endosperm: Nakamura et. al., 1992, Physiologia Plantarum, 84:329–335 and Nakamura and Yamanouchi, 1992, Plant Physiol., 99:1265–1266; pea: Smith, 1988, Planta, 175:270–279 and Bhattacharyya et al., 1989, J. Cell Biochem., Suppl. 13D:331; maize endosperm: Singh and Preiss, 1985, Plant Physiology, 79:34–40; VosScherperkeuter et al., 1989, Plant Physiology, 90:75–84; potato: Kossmann et al., 1991, Mol. Gen. Genet., 230(12):39–44; cassava: Salehuzzaman and Visser, 1992, Plant Mol Biol, 20:809–819).
In the area of polysaccharide enzymes there are reports of vectors for engineering modification in the starch pathway of plants by use of a number of starch synthesis genes in various plant species. That some of these polysaccharide enzymes bind to cellulose or starch or glycogen is well known. One specific patent example of the use of a polysaccharide enzyme shows the use of glycogen biosynthesis enzymes to modify plant starch. In U.S. Pat. No. 5,349,123 to Shewmaker a vector containing DNA to form glycogen biosynthetic enzymes within plant cells is taught. Specifically, this patent refers to the changes in potato starch due to the introduction of these enzymes. Other starch synthesis genes and their use have also been reported.
Hybrid (Fusion) Peptides
Hybrid proteins (also called “fusion proteins”) are polypeptide chains that consist of two or more proteins fused together into a single polypeptide. Often one of the proteins is a ligand which binds to a specific receptor cell. Vectors encoding fusion peptides are primarily used to produce foreign proteins through fermentation of microbes. The fusion proteins produced can then be purified by affinity chromatography. The binding portion of one of the polypeptides is used to attach the hybrid polypeptide to an affinity matrix. For example, fusion proteins can be formed with beta galactosidase which can be bound to a column. This method has been used to form viral antigens.
Another use is to recover one of the polypeptides of the hybrid polypeptide. Chemical and biological methods are known for cleaving the fused peptide. Low pH can be used to cleave the peptides if an acid-labile aspartate-proline linkage is employed between the peptides and the peptides are not affected by the acid. Hormones have been cleaved with cyanobromide. Additionally, cleavage by site-specific proteolysis has been reported. Other methods of protein purification such as ion chromatography have been enhanced with the use of polyarginine tails which increase the overall basicity of the protein thus enhancing binding to ion exchange columns.
A number of patents have outlined improvements in methods of making hybrid peptides or specific hybrid peptides targeted for specific uses. U.S. Pat. No. 5,635,599 to Pastan et al. outlines an improvement of hybrid proteins. This patent reports a circularly permuted ligand as part of the hybrid peptide. This ligand possesses specificity and good binding affinity. Another improvement in hybrid proteins is reported in U.S. Pat. No. 5,648,244 to Kuliopulos. This patent describes a method for producing a hybrid peptide with a carrier peptide. This nucleic acid region, when recognized by a restriction endonuclease, creates a nonpalindromic 3-base overhang. This allows the vector to be cleaved.
An example of a specifically targeted hybrid protein is reported in U.S. Pat. No. 5,643,756. This patent reports a vector for expression of glycosylated proteins in cells. This hybrid protein is adapted for use in proper immunoreactivity of HIV gp120. The isolation of gp120 domains which are highly glycosylated is enhanced by this reported vector.
U.S. Pat. Nos. 5,202,247 and 5,137,819 discuss hybrid proteins having polysaccharide binding domains and methods and compositions for preparation of hybrid proteins which are capable of binding to a polysaccharide matrix. U.S. Pat. No. 5,202,247 specifically teaches a hybrid protein linking a cellulase binding region to a peptide of interest. The patent specifies that the hybrid protein can be purified after expression in a bacterial host by affinity chromatography on cellulose.
The development of genetic engineering techniques has made it possible to transfer genes from various organisms and plants into other organisms or plants. Although starch has been altered by transformation and mutagenesis in the past there is still a need for further starch modification. To this end vectors that provide for encapsulation of desired amino acids or peptides within the starch and specifically within the starch granule are desirable. The resultant starch is modified and the tissue from the plant carrying the vector is modified.